Broth versus solid agar culture of swab samples of cadaveric allograft musculoskeletal tissue

As part of the donor assessment protocol, bioburden assessment must be performed on allograft musculoskeletal tissue samples collected at the time of tissue retrieval. Swab samples of musculoskeletal tissue allografts from cadaveric donors are received at the microbiology department of the South Eastern Area Laboratory Services (Australia) to determine the presence of bacteria and fungi. This study will review the isolation rate of organisms from solid agar and broth culture of swab samples of cadaveric allograft musculoskeletal tissue over a 6-year period, 2006–2011. Swabs were inoculated onto horse blood agar (anaerobic, 35 °C) and chocolate agar (CO₂, 35 °C) and then placed into a cooked meat broth (aerobic, 35 °C). A total of 1,912 swabs from 389 donors were received during the study period. 557 (29.1 %) swabs were culture positive with the isolation of 713 organisms, 249 (34.9 %) from solid agar culture and an additional 464 (65.1 %) from broth culture only. This study has shown that the broth culture of cadaveric allograft musculoskeletal swab samples recovered a greater amount of organisms than solid agar culture. Isolates such as Clostridium species and Staphylococcus aureus would not have been isolated from solid agar culture alone. Broth culture is an essential part of the bioburden assessment protocol of swab samples of cadaveric allograft musculoskeletal tissue in this laboratory.     

Swab or biopsy samples for bioburden testing of allograft musculoskeletal tissue?

Swab and biopsy samples of allograft musculoskeletal tissue are most commonly collected by tissue banks for bacterial and fungal bioburden testing. An in vitro study was performed using the National Committee for Clinical Laboratory Standards standard ‘Quality control of microbiological transport systems’ (2003) to validate and evaluate the recovery of six challenge organisms from swab and biopsy samples of allograft musculoskeletal tissue. On average, 8.4 to >100 and 7.2 to >100 % of the inoculum was recovered from swab and biopsy samples respectively. A retrospective review of donor episodes was also performed, consisting of paired swab and biopsy samples received in this laboratory during the period 2001–2012. Samples of allograft femoral heads were collected from living donors during hip operations. From the 3,859 donor episodes received, 21 paired swab and biopsy samples each recovered an isolate, 247 swab samples only and 79 biopsy samples only were culture positive. Low numbers of challenge organisms were recovered from inoculated swab and biopsy samples in the in vitro study and validated their use for bioburden testing of allograft musculoskeletal tissue. Skin commensals were the most common group of organisms isolated during a 12-year retrospective review of paired swab and biopsy samples from living donor allograft femoral heads. Paired swab and biopsy samples are a suitable representative sample of allograft musculoskeletal tissue for bioburden testing.     

Fungal culture of musculoskeletal tissue: what’s the point?

There have not been any studies that review the prevalence of fungal isolates using selective media from samples of banked musculoskeletal tissue retrieved from living and cadaveric donors. A total of 2,036 swab and 2,621 biopsy samples of musculoskeletal tissue from tissue banks were received from the 1st August 2008 till 31st December 2010. Routine culture for fungi using selective media with a prolonged incubation period failed to demonstrate a greater prevalence of fungal isolates than by using non-selective culture media alone. Using selective culture fungi were recovered from only two Sabouraud agar plates (0.1%) but not from non-selective media. During the same period fungi were isolated from three graft samples cultured in non-selective broth media only (0.1%). There was no correlation of fungal isolates from selective or non-selective media inoculated at the same time nor from multiple graft samples collected from the same donor supporting the possibility of an exogenous source for fungal isolates rather than an endogenous source.     

Assessment of bioburden on human and animal tissues: Part 2—Results of testing of human tissue and qualification of a composite sample for routine bioburden determination

A quantitative method was developed and validated to assess bioburden on tissue from human donors and to compare bioburden determination results to swab culture results from the same donor. An initial study with allograft tissue from 101 donors showed a wide range of bioburden levels; values from no colony-forming units (CFU) detected to?>28,000?CFU were observed. Tissues from donors that had swab cultures negative for objectionable microorganisms generally had lower bioburden than tissues from donors where objectionable microorganisms were recovered by swab culturing. In a follow-up study with 1,445 donors, a wide range of bioburden levels was again observed on tissues from donors that were swab culture negative for objectionable microorganisms. Tissues from 885 (61%) of these donors had no recoverable bioburden (<2?CFU). Importantly, tissues from 560 (39%) of the donors had recoverable bioburden which ranged from 1 to?>24,000?CFU. Identification of bioburden isolates showed a diversity of genera and species. In compliance with the recent revision of the American Association of Tissue Banks K2.210 Standard, the quantitative bioburden determination method was validated with a composite tissue sample that contains bone and soft tissue sections tested together in one extraction vessel. A recovery efficiency of 68% was validated and the composite sample was shown to be representative of all of the tissues recovered from a donor. The use of the composite sample in conjunction with the quantitative bioburden determination method will facilitate an accurate assessment of the numbers and types of contaminating microorganisms on allografts prior to disinfection/sterilization. This information will ensure that disinfection/sterilization processes are properly validated and the capability of the overall allograft process is understood on a donor by donor basis.     

Analysis of the effectiveness of Sodium Hypochlorite decontamination of cadaveric human tissues at retrieval

Bacterial contamination of tissues retrieved from cadaveric donors is a common feature worldwide, and every tissue bank, albeit using different methods, conducts decontamination to guarantee safe tissues suitable for clinical use. The effectiveness of the methods used to eradicate pathogens differs. In order to reduce the tissue bioburden at retrieval, we have introduced a new method involving rinsing tissues in a sodium hypochlorite solution. To test its effectiveness we analyzed two comparable groups of tissues: Group A: 1881 tissues, all rinsed with isotonic saline solution after retrieval, and Group B: 1968 tissues immersed in an isotonic saline solution containing sodium hypochlorite (final concentration 0.1 %) for different lengths of time and subsequently rinsed with isotonic saline. The rinsing solution of each tissue was then sampled for microbiological cultures in both groups. The resultant overall contamination rate was 40.5 % for Group A and 6.7 % for Group B, with an 82.8 % difference in the reduction of contamination between the two groups. This was especially the case for commensal skin bacteria in musculoskeletal tissue, which accounted for over half the overall contamination. Our data highlighted that decontamination with sodium hypochlorite was helpful in reducing the bacterial bioburden in tissues retrieved from cadaveric donors.     

The visual assessment of broth cultures for tissue bank samples

The bioburden screening process of allograft musculoskeletal tissue samples received at the South Eastern Area Laboratory Services includes the routine use of solid agar and cooked meat (CM) broth media. CM has been routinely sub-cultured onto solid agar plates after aerobic incubation at 35 °C. This study will evaluate whether a visual assessment of CM can replace sub-culture by an in vitro inoculation and a prospective study. Eight challenge organisms were serially diluted and inoculated into CM. The average inoculum of 0.5–5.5 CFU produced visible turbidity of CM after 24-h incubation for 7 of the challenge organisms with one organism producing turbidity after 48-h incubation. The prospective study evaluated 222 CM of which 213 were visually clear and no-growth on sub-culture and 9 turbid CM which were culture positive. Broth cultures are an integral part of the bioburden screening process of allograft musculoskeletal tissue and swab samples and visual assessment of CM can replace sub-culture.     

Bacterial Contamination of Tissue Allografts – Experiences of the Donor Tissue Bank of Victoria

The aim of this study is to report the experience of the Donor Tissue Bank of Victoria with bacteria isolated from musculoskeletal, skin and cardiac allografts retrieved from cadaveric donors. The results of all quality control samples for bacterial culture, taken during retrieval and processing of allografts at the DTBV for a 12 month period, were extracted and analysed. It was found that 15.7% of skin, 15.1% of heart valves and 5.8% of musculoskeletal samples had positive culture results. The number and types of organisms isolated varied with tissue type. The most commonly isolated organisms were Staphylococcus species (including S. aureus). The identity of the isolate and the number of positive specimens from the same donor were considerations in the decision concerning the suitability of tissue for subsequent implantation.     

Bioburden in transport solutions of human cardiovascular tissues: a comparative evaluation of direct inoculation and membrane filter technique

All cardiac allograft tissues are under potential contamination, requiring a validated terminal sterilization process or a minimal bioburden. The bioburden calculation is important to determine the bacterial burden and further decontamination and disinfection strategies for the valve processing. The aim of this study was to determine the bioburden from transport solution (TS) of heart valves obtained from non-heart-beating and heart-beating donors in different culture methods. The bioburden from TS was determined in 20 hearts donated for valve allograft tissue using membrane filter (MF) and direct inoculation. Tryptic soy agar and Sabouraud plates were incubated and colonies were counted. Ninety-five percent of samples from this study were obtained from heart-beating donors. The warm ischemic time period for heart was 1.06?±?0.74 h and the cold ischemic time period was 25.66?±?11.16 h. The mean TS volume was 232.68?±?96.67 mL (48.5–550 mL). From 20 samples directly inoculated on TSA agar plates, 2 (10%) were positive. However, when MF was used, from 20 samples in TSA, 13 (65%) were positive with a mean count of 1.36?±?4.04 CFU/mL. In Sabouraud plates, the direct inoculation was positive in 5 samples (25%) with a mean count of 0.24?±?0.56 CFU/mL. The use of MF increased the positivity to 50% (10 samples from a total of 20) with a mean of 0.28?±?0.68 CFU/mL. The positivity was superior using MF in comparison with direct inoculation (p?<?0.05). The bioburden of TS is low and MF is the technique of choice due to higher positivity.     

Analysis of Potential Causes of Positive Microbiological Cultures in Tissue Donors

The purpose of this statistical analysis is to determine what factors are the major contributors to bacterial contamination of recovered human cadaveric tissue. In this study we analyzed factors that could contribute to an increased bacterial bioburden from recovered tissues using the following independent variables: (1) the physical recovery environment; (2) recovery before or after an autopsy; (3) the length of time from death to recovery; (4) the cause of death; (5) the length of time to complete recovery; (6) the number of staff involved with the tissue recovery; and (7) the impact of organ and skin recovery on musculoskeletal contamination rates.In these analyses we used analysis of variance of main effects on data from seven tissue banks. The scale of the analysis included 1036 donors each having multiple cultures to better control for the inherent large variation in this type of data. We looked at several dependent variables. The dependent variable that was most useful was percent positive cultures.The results of the combined data differed from analyzing the tissue banks individually. The differences in each tissue bank's procedures and techniques were responsible for most of the variability. Depending on how the data was organized, statistically significant increases in bioburden were seen with: (1) recoveries after autopsy; (2) location of the recovery; (3) length of time taken for a recovery; (4) size of the recovery team; and (5) the impact of organ and skin recovery on musculoskeletal contamination rates.In conclusion, statistical analysis of recovery cultures can be a powerful tool that may be used to indicate problems within any bank's recovery procedures or techniques.     

Culture methods of allograft musculoskeletal tissue samples in Australian bacteriology laboratories

Samples of allograft musculoskeletal tissue are cultured by bacteriology laboratories to determine the presence of bacteria and fungi. In Australia, this testing is performed by 6 TGA-licensed clinical bacteriology laboratories with samples received from 10 tissue banks. Culture methods of swab and tissue samples employ a combination of solid agar and/or broth media to enhance micro-organism growth and maximise recovery. All six Australian laboratories receive Amies transport swabs and, except for one laboratory, a corresponding biopsy sample for testing. Three of the 6 laboratories culture at least one allograft sample directly onto solid agar. Only one laboratory did not use a broth culture for any sample received. An international literature review found that a similar combination of musculoskeletal tissue samples were cultured onto solid agar and/or broth media. Although variations of allograft musculoskeletal tissue samples, culture media and methods are used in Australian and international bacteriology laboratories, validation studies and method evaluations have challenged and supported their use in recovering fungi and aerobic and anaerobic bacteria.     

Molecular testing of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> contaminating tissue allografts recovered from deceased donors

Microbiological screening of tissue allografts is crucial to prevent the transmission of bacterial and fungal infections to transplant recipients. Klebsiella was the most prevalent and resistant contaminating microorganism observed in our setting in the Iranian Tissue Bank. This study was conducted to determine the presence of extended-spectrum β-lactamase (ESBL) genes, antimicrobial resistance patterns of Klebsiella pneumoniae isolates, and their clonal relationships in allograft materials. K. pneumoniae contaminating bone and other tissue allografts recovered from deceased donors were identified and ESBL isolates were detected using a phenotypic confirmatory method. Antimicrobial susceptibility testing was carried out using the disk diffusion method. Distribution of ESBL genes and molecular typing were performed using polymerase chain reaction (PCR) and Repetitive-element (rep-PCR) methods. Of 3828 donated tissues, 51 (1.3%) were found contaminated by K. pneumoniae isolates. Compared to tissue allografts from brain-dead, heart-beating tissue donors, allografts from donors with circulatory cessation were associated with a higher risk of K. pneumoniae contamination [odds ratio (OR), 1.2 (CI 95% 0.9–2.3) (P value < 0.001)]. Half of the isolates produced ESBL, and the rate of susceptibility to cephalosporins was 51%. Among isolates, 22 (43.1%) harbored CTX-M, 31 (60.8%) SHV, and 9 (17.6%) harbored TEM types. The rep-dendrogram indicated that clones having identical or related strains with a similar antibiotype were isolated in the same period. This study provides evidence that a single clone of K. pneumoniae contaminated tissue allografts recovered from many different donors. A single clone found on tissues from several donors suggests contamination of tissues from a single source such as the tissue recovery process and environment. Genomic DNA testing and clonality of contaminating bacteria using molecular methods can focus the epidemiologic investigation on the tissue allograft recovery process including a search for contamination of the tissue recovery room environment, recovery staff, recovery equipment, reagents, solutions and supplies.     

Infection not associated with use of human musculoskeletal tissue allografts

The deaths of otherwise healthy patients that are attributable to contaminated allografts have heightened concerns about the screening, processing, and use of such tissues. We present one tertiary care institution’s experience with musculoskeletal allografts and determine the frequency of postoperative Clostridium infection. We used an institutional microbiology database to identify all records of culture-confirmed Clostridium infection from January 1990 through July 2006. A comprehensive musculoskeletal database was cross-referenced to include all possible allograft samples surgically collected or implanted from January 1990 through July 2004 to determine the frequency of Clostridium infection associated with use of allograft musculoskeletal tissue. Musculoskeletal allografts were implanted in 16,314 patients during the study period. After a minimum follow-up of 2 years, no patient had development of a definite Clostridium infection that was attributable to the use of musculoskeletal allograft tissue. These outcomes can be achieved with established screening and processing techniques for donor tissue.     

Parasitic Bacteria and Fungi on Common Mistletoe (Viscum album L.) and Their Potential Application in Biocontrol

This study was carried out to identify pathogenic bacteria and fungi on mistletoe (Viscum album L.) and investigate their potential use in biological control of this parasitic plant. For this purpose, a total of 48 fungal isolate and 193 bacterial strains were isolated from contaminated V. album during the summers 2005–2006. The isolated bacterial strains and fungal isolates were identified by using the Sherlock Microbial Identification System (MIS; Microbial ID, Newark) and microscopic methods, respectively. The bacterial strains that induced hypersensitive reaction (HR) on tobacco (Nicotiana tabacum L.) and fungal isolates were tested for pathogenicity on young shoots of mistletoe by using injection methods. The pathogenic bacterial strains and fungal isolates were also tested for their activity against mistletoe using spray methods. Five bacterial strains (two Burkholderia cepacia, one each of Bacillus megaterium, Bacillus pumilus and Pandoraea pulminicola) were HR and pathogenicity positive when injected but none of them when sprayed on mistletoe. When fungi were injected, 32 isolates were pathogenic but only thirteen when sprayed on mistletoe. Alternaria alternata VA?‐202, VA?‐205, VA?‐217 and Acremonium kiliense VA‐11 fungal isolates were the most effective ones and caused strong disease symptoms on mistletoe. The present study is the first report on the efficiency of potential biocontrol agents against mistletoe in Turkey.     

Bioburden assessment of banked bone used for allografts

Allograft bone is commonly used in reconstructive orthopaedic surgery and needs to be assessed for bioburden before transplant. The Microbiology Department of the South Eastern Area Laboratory Services (SEALS), located at the St. George Hospital, Sydney, has provided this service to the New South Wales (NSW) Bone Bank. This study reviewed the organisms isolated from femoral head allografts of living donors from the NSW Bone Bank over a 7-year period. It was found that growth was reported from 4.9% of samples with the predominant organism being coagulase-negative staphylococci. This review will focus on the micro-organisms isolated, the interaction of the laboratory with the bone bank, the relevance of the bioburden assessment in the overall quality process and patient safety.     

Disinfection of human musculoskeletal allografts in tissue banking: a systematic review

Musculoskeletal allografts are typically disinfected using antibiotics, irradiation or chemical methods but protocols vary significantly between tissue banks. It is likely that different disinfection protocols will not have the same level of microorganism kill; they may also have varying effects on the structural integrity of the tissue, which could lead to significant differences in terms of clinical outcome in recipients. Ideally, a disinfection protocol should achieve the greatest bioburden reduction with the lowest possible impact on tissue integrity. A systematic review of three databases found 68 laboratory and clinical studies that analyzed the microbial bioburden or contamination rates of musculoskeletal allografts. The use of peracetic acid–ethanol or ionizing radiation was found to be most effective for disinfection of tissues. The use of irradiation is the most frequently published method for the terminal sterilization of musculoskeletal allografts; it is widely used and its efficacy is well documented in the literature. However, effective disinfection results were still observed using the BioCleanse? Tissue Sterilization process, pulsatile lavage with antibiotics, ethylene oxide, and chlorhexidine. The variety of effective methods to reduce contamination rate or bioburden, in conjunction with limited high quality evidence provides little support for the recommendation of a single bioburden reduction method.     

Co-culture of dedifferentiated and primary human chondrocytes obtained from cadaveric donor enhance the histological quality of repair tissue: an in-vivo animal study

To compare the quality of the repair tissue in three-dimensional co-culture of human chondrocytes implanted in an in vivo model. Six cadaveric and five live human donors were included. Osteochondral biopsies from the donor knees were harvested for chondrocyte isolation. Fifty percent of cadaveric chondrocytes were expanded until passage-2 (P2) while the remaining cells were cryopreserved in passage-0 (P0). Fresh primary chondrocytes (P0f) obtained from live human donors were co-cultured. Three-dimensional constructs were prepared with a monolayer of passage-2 chondrocytes, collagen membrane (Geistlich Bio-Gide^®), and pellet of non-co-cultured (P2) or co-cultured chondrocytes (P2 + P0c, P2 + P0f). Constructs were implanted in the subcutaneous tissue of athymic mice and left for 3 months growth. Safranin-O and Alcian blue staining were used to glycosaminoglycan content assessment. Aggrecan and type-II collagen were evaluated by immunohistochemistry. New-formed tissue quality was evaluated with an adaptation of the modified O’Driscoll score. Histological quality of non-co-cultured group was 4.37 (SD ±4.71), while co-cultured groups had a mean score of 8.71 (SD ±3.98) for the fresh primary chondrocytes and 9.57 (SD ±1.27) in the cryopreserved chondrocytes. In immunohistochemistry, Co-culture groups were strongly stained for type-II and aggrecan not seen in the non-co-cultured group. It is possible to isolate viable chondrocytes from cadaveric human donors in samples processed in the first 48-h of dead. There is non-significant difference between the numbers of chondrocytes isolated from live or cadaveric donors. Cryopreservation of cadaveric primary chondrocytes does not alter the capability to form cartilage like tissue. Co-culture of primary and passaged chondrocytes enhances the histological quality of new-formed tissue compared to non-co-cultured cells.     

Bacterial infection transmitted by human tissue allograft transplantation

Bacterial contamination of tissue allografts obtained from cadaveric donors has been a serious cause of morbidity and mortality in recipients. Recent cases of fatal and nonfatal bacterial infections in recipients of contaminated articular cartilage (distal femur) and tendon allografts have called attention to the importance of avoiding tissue donors suspected of carrying infectious disease, of not processing donated tissue carrying virulent bacteria, the occurrence of falsely negative final sterility tests, and the need to sterilize tissues. These cases demonstrated that contamination can arise from an infected donor, during tissue removal from cadaveric donors, from the processing environment, and from contaminated supplies and reagents used during processing. Final sterility testing can be unreliable, especially when antibiotics remain on tissues. There is an increasing need for control of microbial contamination in tissue banks, and sterilization of tissue allografts should be recommended whenever possible.     

Supply of human allograft tissue in Canada

There is relatively little known about the supply for allograft tissues in Canada. The major aim of this study is to quantify the current or “Known Supply” of human allograft tissue (bone, tendons, soft tissue, cardiovascular, ocular and skin) from known tissue banks in Canada, to estimate the “Unknown Supply” of human allograft tissue available to Canadian users from other sources, and to investigate the nature and source of these tissue products. Two surveys were developed; one for tissue banks processing one or more tissue types and the other specific to eye banks. Thirty nine sites were initially identified as potential tissue bank respondent sites. Of the 39 sites, 29 sites indicated that they were interested in participating or would consider completing the survey. A survey package and a self-addressed courier envelope were couriered to each of 29 sites. A three week response time was indicated. The project consultants conducted telephone and email follow-up for incomplete data. Unknown supply was estimated by 5 methods. Twenty-eight of 29 sites (97%) completed and returned surveys. Over the past year, respondents reported a total of 5,691 donors (1,550 living and 4,141 cadaveric donors). Including cancellous ground bone, there were 10,729 tissue products produced by the respondent banks. Of these, 71% were produced by accredited banks and 32% were ocular tissues. Total predicted shortfall of allograft tissues was 31,860–66,481 grafts. Through estimating Current supply, and compiling additional qualitative information, this study has provided a snapshot of the current Canadian supply and shortfall of allograft tissue grafts.     

Organogenesis forum lecture

Renal replacement therapy (i.e., kidney transplantation) represents the optimal treatment for end-stage renal disease (a condition which is expected to increase over the next decade or so). However, the demand for transplantable kidneys currently outpaces the availability of donor kidneys, a situation not expected to improve in the foreseeable future. An alternative route to cadaveric or living-related donors would be to engineer kidneys for allograft transplantation from cells based on concepts derived from our current understanding of normal kidney development. Although the use of cells for this purpose remains hypothetical, recent research from our laboratory has provided strong evidence that implantation of kidney-like tissue bio-engineered from the recombination of in vitro culture systems which model discrete aspects of kidney development (i.e., cell culture, isolated WD, isolated UB, and isolated MM) is possible. These recent findings are discussed here. Pathway based system biology approaches to understanding the mechanism(s) of kidney development are also discussed, particularly in the setting of this novel and seemingly powerful xeno-based tissue engineering strategy.     

Rhizobacteria of Cotton and Their Repression of Seedling Disease Pathogens

During the 1983 field season, the rhizobacteria (including organisms from rhizosphere soil and the root rhizoplane) of cotton plants at one location in Mississippi were inventoried at different plant growth stages. Isolates (1,000) were identified to the genus level and characterized for repression of Pythium ultimum and Rhizoctonia solani. Cotton seedlings were initially colonized by bacteria of many different genera, and populations quickly reached 10⁸ CFU/g of root tissue. As the season progressed, the bacterial populations declined as root mass increased and the roots became more woodlike in consistency. Fluorescent pseudomonads were the most numerous gram-negative rhizobacterial isolates of those that were randomly collected and identified, and they provided the largest number of isolates with fungal repressive activity. Several other gram-negative bacterial genera were recovered throughout the growing season, and some gram-positive bacteria were also isolated routinely, but at lower numbers. There was no correlation between the proportion of rhizobacterial isolates that possessed fungal repressive activity and the plant growth stage from which the isolates were obtained. Approximately twice as many bacterial isolates demonstrated fungal repression in the agar assay compared with the inplanta assay, and isolates were found more frequently with fungal repressive activity against P. ultimum than against R. solai.